Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with caborundum
Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with caborundum
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DOI：
10.1007/bf01890852
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发表时间：
1996-12-01
影响因子：
6.2
通讯作者：
Yeh, SD
中科院分区：
文献类型：
--
作者：
Cheng, YH;Yang, JS;Yeh, SD
转基因番木瓜（Carica papaya L.）由于常规农杆菌感染或微粒轰击获得的转化率低，我们描述了一种高效的农杆菌介导的转化方法的基础上培养的胚性组织与卡波姆在液相中的创伤。从授粉后75-90天收集的培养的未成熟合子胚获得胚发生组织。将番木瓜环斑病毒（PRSV）台湾株系的可表达外壳蛋白（CP）基因构建到Ti双元载体pBGCP中，该载体含有NPT-Ⅱ基因作为选择标记。将胚发生组织与600目卡波姆在无菌蒸馏水中涡旋1分钟，然后用去武装的A处理。含有pBGCP的根瘤菌。转化细胞在含有2，4-D和羧苄青霉素的无卡那霉素培养基上培养2-3周，然后在卡那霉素培养基上培养3-4个月。将发育好的体细胞胚转移到含有NAA、BA和卡那霉素的培养基上，然后再生出外观正常的植株。通过PCR检测推定的转基因株系中PRSV CP基因的存在，并通过Western印迹验证CP的表达。在回交的R-1后代中分离分析表明，转基因是核遗传的。从5个独立的实验中，平均转化成功率为15.9%的合子胚处理（52转基因体细胞胚簇的合子胚处理的327），约10-100倍，比以前报道的方法。因此，通过碳纤维涡旋法损伤可再生性强的分化组织，为农杆菌介导的番木瓜遗传转化提供了一种简单有效的途径。
Generation of transgenic papaya (Carica papaya L.) has been hampered by the low rates of transformation achieved by conventional Agrobacterium infection or microprojectile bombardment. We describe an efficient Agrobacterium-mediated transformation method based on wounding of cultured embryogenic tissues with carborundum in liquid phase. Embryogenic tissues were obtained from cultured immature zygotic embryos collected 75-90 days after pollination. The expressible coat protein (CP) gene of a Taiwan strain of papaya ringspot virus (PRSV) was constructed in a Ti binary vector pBGCP, which contained the NPT-II gene as a selection marker. The embryogenic tissues were vortexed with 600 mesh carborundum in sterile distilled water for 1 min before treating with the disarmed A. tumefaciens containing the pBGCP. Transformed cells were cultured on kanamycin-free medium containing 2,4-D and carbenicillin for 2-3 weeks and then on the kanamycin medium for 3-4 months. The developed somatic embryos were transferred to the medium containing NAA, BA and kanamycin and subsequently regenerated into normal-appearing plants. Presence of the PRSV CP gene in the putative transgenic lines was detected by PCR and the expression of the CP was verified by Western blotting. The transgene was nuclearly inherited as revealed by segregation analysis in the backcrossed R-1 progeny. From five independent experiments, the average successful rate of transformation was 15.9% of the zygotic embryos treated (52 transgenic somatic embryo clusters out of 327 zygotic embryos treated), about 10-100 times higher than the available methods previously reported. Thus, wounding highly regenerable differentiating tissues by carborundum vortexing provides a simple and efficient way for papaya transformation mediated by Agrobacterium.